Therapeutic hypothermia in neonatal hypoxic encephalopathy: A systematic review and meta-analysis

Background Therapeutic hypothermia (TH) is regarded as the most efficacious therapy for neonatal hypoxic encephalopathy. However, limitations in previous systematic reviews and the publication of new data necessitate updating the evidence. We conducted this up-to-date systematic review to evaluate the effects of TH in neonatal encephalopathy on clinical outcomes. Methods In this systematic review and meta-analysis, we searched Medline, Cochrane Library, Embase, LIVIVO, Web of Science, Scopus, CINAHL, major trial registries, and grey literature (from inception to October 31, 2021), for randomized controlled trials (RCT) comparing TH vs normothermia in neonatal encephalopathy. We included RCTs enrolling neonates (gestation ≥35 weeks) with perinatal asphyxia and encephalopathy, who received either TH (temperature ≤34°C) initiated within 6 hours of birth for ≥48 hours, vs no cooling. We excluded non-RCTs, those with delayed cooling, or cooling to >34°C. Two authors independently appraised risk-of-bias and extracted data on mortality and neurologic disability at four time points: neonatal (from randomization to discharge/death), infancy (18-24 months), childhood (5-10 years), and long-term (>10 years). Other outcomes included seizures, EEG abnormalities, and MRI findings. Summary data from published RCTs were pooled through fixed-effect meta-analysis. Results We identified 36 863 citations and included 39 publications representing 29 RCTs with 2926 participants. Thirteen studies each had low, moderate, and high risk-of-bias. The pooled risk ratios (95% confidence interval, CI) were as follows: neonatal mortality: 0.87 (95% CI = 0.75, 1.00), n = 2434, I2 = 38%; mortality at 18-24 months: 0.88 (95% CI = 0.78, 1.01), n = 2042, I2 = 51%; mortality at 5-10 years: 0.81 (95% CI = 0.62, 1.04), n = 515, I2 = 59%; disability at 18-24 months: 0.62 (95% CI = 0.52, 0.75), n = 1440, I2 = 26%; disability at 5-10 years: 0.68 (95% CI = 0.52, 0.90), n = 442, I2 = 3%; mortality or disability at 18-24 months: 0.78 (95% CI = 0.72, 0.86), n = 1914, I2 = 54%; cerebral palsy at 18-24 months: 0.63 (95% CI = 0.50, 0.78), n = 1136, I2 = 39%; and childhood cerebral palsy: 0.63 (95% CI = 0.46, 0.85), n = 449, I2 = 0%. Some outcomes showed significant differences by study-setting; the risk ratio (95% CI) for mortality at 18-24 months was 0.79 (95% CI = 0.66,0.93), n = 1212, I2 = 7% in high-income countries, 0.67 (95% CI = 0.41, 1.09), n = 276, I2 = 0% in upper-middle-income countries, and 1.18 (95% CI = 0.94, 1.47), n = 554, I2 = 75% in lower-middle-income countries. The corresponding pooled risk ratios for ‘mortality or disability at 18-24 months’ were 0.77 (95% CI = 0.69, 0.86), n = 1089, I2 = 0%; 0.56 (95% CI = 0.41, 0.78), n = 276, I2 = 30%; and 0.92 (95% CI = 0.77, 1.09), n = 549, I2 = 86% respectively. Trials with low risk of bias showed risk ratio of 0.97 (95% CI = 0.80, 1.16, n = 1475, I2 = 62%) for neonatal mortality, whereas trials with higher risk of bias showed 0.71 (95% CI = 0.55, 0.91), n = 959, I2 = 0%. Likewise, risk ratio for mortality at 18-24 months was 0.96 (95% CI = 0.83, 1.13), n = 1336, I2 = 58% among low risk-of-bias trials, but 0.72 (95% CI = 0.56, 0.92), n = 706, I2 = 0%, among higher risk of bias trials. Conclusions Therapeutic hypothermia for neonatal encephalopathy reduces neurologic disability and cerebral palsy, but its effect on neonatal, infantile and childhood mortality is uncertain. The setting where it is implemented affects the outcomes. Low(er) quality trials overestimated the potential benefit of TH.

Neonatal hypoxic ischemic encephalopathy is a significant cause of mortality and morbidity. It is also associated with adverse outcomes such as cerebral palsy, cognitive dysfunction, epilepsy, and others, well beyond the neonatal period. These have a cascading impact on the community and society through increased health care utilization, need for special services, economic burden, and diminished workforce productivity. Several interventions have been explored to manage neonatal encephalopathy (NE). Among these, therapeutic hypothermia (TH) is ranked highest, with several studies and systematic reviews [1,2] reporting reduction in mortality and adverse neurological and/or neurodevelopmental outcomes during infancy [3,4]. TH involves controlled cooling of the body (or at least of the head) during the first 2-4 days of life, followed by a gradual rewarming to a euthermic state [1,5]. Currently, it is implemented globally, including in many low-resource health care settings [6][7][8], although the International Liaison Committee on Resuscitation advised its use only in institutions with adequate monitoring and intensive care facilities [9].
A recent multi-country HELIX trial reported that TH was associated with an alarming increase in both immediate and late mortality, prompting the authors to emphatically recommend its immediate discontinuation in resource-constrained settings [10]. This created considerable consternation, especially in some developing countries, with arguments about the trial methods, generalizability, and other issues [11][12][13][14][15][16][17]. However, critical appraisal of the trial confirmed its validity [18], despite some plausible explanations for the stark differences in key outcomes [19]. Additionally, a systematic review restricted to trials from developing countries reported limited benefit of TH in such settings [20].
These developments necessitate a detailed review of the available evidence. The Cochrane review published in 2013 is outdated, and also contained some data analysis errors, such as combining short-term and long-term outcomes in the same meta-analysis [1]. A more recent review, updated as of mid-2020, contained several errors such as duplication of data from some trials, presenting data from non-existent trials, missing relevant trials, combining short-term and long-term mortality together, and expressing relative risk with negative integers [2]. Therefore, we conducted an up-to-date systematic review of randomized controlled trials (RCTs) to evaluate the effects of therapeutic hypothermia (Intervention), vs normothermia or no hypothermia (Comparison), in neonates with hypoxic encephalopathy (Population), on mortality and neurological and/or neurodevelopmental features (Outcomes). The question of this review was: What are the effects of therapeutic hypothermia in newborns with hypoxic encephalopathy?

METHODS
This review was registered in PROSPERO (Registration number CRD42021279682, dated 20 October 2021) [21] and conducted in accordance with the Cochrane Handbook for systematic reviews [22]. The review is reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses-Protocols (PRIS-MA-P) 2020 statement [23].

Types of intervention:
We included RCTs delivering TH (whole-body cooling [WBC] or selective head cooling [SHC]) by any device/equipment, initiated within 6 hours of birth, with documented reduction in core temperature (to ≤34°C in case of WBC) or middle ear temperature (to ≤34°C in case of SHC). We excluded trials where TH was initiated later than six hours after birth (in all or the majority of infants), or cooling was conducted without documentation of core temperature (as specified above), or was done for <48 hours.

Types of comparison:
The comparator was normothermia, or no therapeutic cooling, or no intervention. We excluded studies without a comparison group, those in which the comparison group had received any cooling for any duration, or a historic comparison group.
Types of outcome measures: We considered the following outcomes: mortality, neurological impairment or disability (defined by any standard criteria), the composite outcome of mortality or disability, and cerebral palsy. We assessed these at four time points after randomization: a) Neonatal, ie, from randomization to discharge or death during the initial hospitalization; b) Infancy, ie, at the age of 18-24 months, c) Childhood, ie, at the age of 5-10 years, and d) Long-term, ie, beyond the age of 10 years. Other outcomes were seizures, electroencephalogram (aEEG) abnormalities, MRI findings suggesting neuronal damage during the initial hospitalization, duration of hospitalization, and quality of life. For this analysis, the primary outcome was listed as "mortality or neurological disability" at ≥18 months of age [21].
Information sources: Two authors independently searched the following databases: Medline, Embase, Cochrane Library, LIVIVO, Web of Science, Scopus, and CINAHL. We searched the following clinical trial registries: World Health Organization International Clinical Trials Registry Platform, ClinicalTrials.gov, and Clinical Trials Registry -India. We also hand-searched reference lists of included trials, as well as previous (narrative and systematic) reviews. In addition, we conducted a grey literature search using OpenGrey (www.opengrey. eu/), ProQuest, and Google Scholar. Each database was searched from its date of inception to October 31, 2021, without restrictions based on language or geography.
Search strategy: We used combinations of MeSH terms and synonyms of the following keywords, and their variations: neonate, newborn, perinatal, infant, hypothermia, therapeutic hypothermia, cool, cooling, therapeutic cooling, asphyxia, hypoxia, hypoxic-ischemic, encephalopathy, neonatal encephalopathy. The searches were pilot-tested before finalizing the strategy. The search strategy in representative databases is summarized in Table S1 in the Online Supplementary Document. Selection of studies: Two review authors independently screened citation titles, followed by the abstracts of short-listed citations, followed by full-text of potentially eligible studies (and those without abstracts). Thereafter, two authors independently examined the full text versions of short-listed studies, to confirm eligibility for inclusion, and recorded reasons for exclusion of ineligible studies. Disagreements were discussed and resolved by consensus. After eliminating duplicate publications, a final list of studies was prepared. A PRISMA flow diagram was created, summarizing the search results and process of including studies.
Translation of languages other than English: Non-English publication abstracts were translated using opensource software; if eligible, the full text was translated as well.
Data extraction: Two review authors independently extracted the following information from the included studies. • Trial characteristics: design, study duration, setting, date of publication. • Participant characteristics: inclusion criteria, exclusion criteria, gestational age, birth weight, definition of perinatal asphyxia, definition and severity of encephalopathy, sample size. • Intervention characteristics: WBC or SHC, method of cooling, temperature targeted, method of determining target temperature, cooling duration, cooling cessation criteria. • Comparison characteristics: Temperature targeted, method of determining target temperature, and standard of care. • Outcomes: Data on the outcomes listed above were extracted along with notes/remarks. Dealing with missing data: We attempted to contact the corresponding authors of studies with missing or unclear data.

Data synthesis and statistical analysis:
We presented data on baseline characteristics with descriptive statistics. We pooled data on the outcomes of interest and performed meta-analysis, using Cochrane Review Manager version 5.4 [24]. For dichotomous outcomes, we calculated risk ratios (RR) with 95% confidence interval (CI) using the fixed-effect model. For continuous outcomes, we calculated the weighted mean difference with 95% CI (fixed-effect model). We opted for the fixed-effect model, as the alternative (random effects-model) tends to assign disproportionately greater weight to studies with smaller sample sizes. However, wherever the heterogeneity statistic exceeded 50%, we re-examined the pooled effect with the random effects model also. For data that could not be pooled by meta-analysis, we provided a description, summarizing the key results.
Assessment of methodological quality of included studies: Two authors independently assessed methodological quality, using version 2 of the Cochrane Risk-of-Bias (RoB) tool [25]. We assessed RoB for each reported outcome of each trial, and the overall RoB of each trial.

Subgroup analysis:
We conducted a subgroup analysis based on the following criteria: a) Study setting (defined by the World Bank Classification of the country where the trial was conducted): high-income country (HIC), upper middle-income country (UMIC), lower middle-income country (LMIC), low-income country (LIC); and b) Type of cooling: WBC vs SHC. We planned subgroup analysis based on cooling method (formal devices vs informal methods), but there were insufficient studies.

Sensitivity analysis:
We assessed the impact of low(er) quality studies, by excluding trials with moderate/ high RoB.
In the Jacobs 2011 trial [27], data were missing in 3.6% and 14.5% of survivors in the intervention and comparison groups. The respective proportions in the Li 2009 trial [46] were 17.8% and 6.8%.   Three publications [33,44,56] presented the proportion with neurological disability during childhood, among 442 survivors; pooled RR was 0.68 (95% CI = 0.52, 0.90), I 2 = 3% (Figure 6). The absolute risk difference was -0.12 (95% CI = -0.21, -0.04), I 2 = 0%. The denominators in two of these [33,56] were less than the number of survivors, suggesting missing data. In the third publication [44], the originally randomized number was unavailable. There were no studies reporting the outcome at 10 years of age.

Quality of life
A single trial [37] presented information on quality of life during childhood using various scoring systems. The proportion with Health Utilities Index (HUI3) score was not different in the two arms, RR was 0.76 (95% CI = 0.55, 1.04); and the mean difference of scores was also similar; 0.09 (95% CI = -0.06, 0.23).

Subgroup analysis
We examined the outcomes by study setting ( Table 2). Neonatal mortality and neonatal seizures did not show statistically significant inter-group differences, in any of the four types of countries/settings. TH significantly reduced mortality at 18-24 months in HIC but did not show statistically significant differences in UMIC or LMIC. Similarly, the composite outcome of death or disability at 18-24 months was significantly lowered in HIC and UMIC, but not LMIC. However, neurological disability and cerebral palsy at 18-24 months showed statistically significant reduction across settings.  Subgroup analysis by type of cooling ( Table 3) showed statistically insignificant inter-group differences between WBC and SHC, for mortality (neonatal and at 18-24 months) and seizures at any age. Other outcomes at 18-24 months, namely neurological disability, composite of mortality or disability, and cerebral palsy, were all improved with TH, irrespective of whether the whole body or only the head was cooled.

Sensitivity analysis
Sensitivity analysis excluding trials with moderate/high RoB (from the analysis) did not change the overall result for major clinical outcomes, although the magnitude of effect diminished for some outcomes (Table 4). However, the exclusion changed three statistically significant differences in MRI outcomes to statistically insignificant differences ( Table 4). Examination of pooled risk ratios among trials with low RoB against those with moderate or high RoB showed that TH reduced neonatal mortality and mortality at 18-24 months in trials with moderate/high RoB, but not in trials with low RoB ( Table 4). However neurological disability, cerebral palsy, and the composite outcome of disability or mortality at 18-24 months showed benefit with TH in both types of trials, although the magnitude was less in low RoB trials.

DISCUSSION
This up-to-date systematic review showed that therapeutic hypothermia implemented for neonatal encephalopathy, did not result in statistically significant reductions in mortality during the neonatal period, infancy or later childhood. However, it reduced neurologic disability and cerebral palsy in infancy and childhood, resulting in reduction in the composite outcome of mortality or disability, despite absence of conclusive benefit on mortality alone. EEG abnormalities and multiple MRI outcomes were better in neonates who received TH. However, there was no statistically significant impact on seizures during the neonatal period, infantile epilepsy, or childhood epilepsy.
While the type of cooling (ie, WBC or SHC) did not affect the results, the setting where TH was implemented was relevant. TH reduced mortality at 18-24 months in high income countries, but not in other settings. While neonatal mortality and seizures were not reduced in any setting, disability and cerebral palsy in infancy were reduced in all settings.
More important, reduction in mortality reported in previous systematic reviews [1,2] was influenced by trials with higher risk of bias.
Thus, this systematic review uncovered several novel findings that contradict previous reviews [1,2]. This is partly because of the availability of new trials, notably the HELIX trial [10], but also due to methodological errors in the previous reviews. The Cochrane review combined immediate and later mortality in the same meta-analysis [1]. The later review failed to include some eligible trials, duplicated data from some trials, presented data from non-existent trials, combined immediate and later mortality, and even expressed relative risk with negative integers [2].
The HELIX trial [10] reported increased mortality (neonatal and infancy) with TH, in stark contrast to previous trials. This RCT was one of the best conducted trials with multiple methodological refinements, strict definitions, largest sample size, extremely low attrition rate, and low risk of bias. Extensive critical appraisal did not identify any major limitations [18], although some concerns were raised about the inclusion of out-born infants, slightly delayed initiation of cooling (though within the accepted limit of 6 hours), and possibly diverse causes of hypoxic encephalopathy in low-resource settings [19].
This systematic review had several strengths notably exhaustive literature search across published and grey literature, inclusion of the largest cohort of trials to date, searching and data extraction in duplicate, careful extraction of data meeting the review criteria (rather than including data reported by trials), and undertaking multiple subgroup and sensitivity analyses. There were no deviations from the protocol [21]. In fact, several additional outcomes were also presented. This fosters high confidence in the review findings.
We acknowledge several limitations in our review. We could not search Chinese language databases, or conference proceedings. We could not obtain individual participant data, or missing data for intention-to-treat analyses. In the protocol, we mentioned that randomized controlled trials would be included, but did not specify how quasi or pseudo randomized studies, would be handled. Analysis of the randomization method identified that 18 trials used an appropriate method of randomization, 1 trial used a quasi-randomization method, and 10 trials had an unclear method. Thus, the included trials had some quasi/pseudo randomized studies. The impact of this is evident from the differences in some outcomes among trials with low vs higher RoB.
The effect of therapeutic hypothermia may also be influenced by several factors such as the proportion of outborn neonates in studies, proportion with severe encephalopathy, method of cooling (servo vs non-servo),  mo -months, y -years *All data are presented as risk ratios (RR) with 95% CI. 'N' represents the number of trials, and 'n' represents the number of participants. and severity of asphyxia. For example, 4 trials excluded outborn neonates, 14 trials included them (but only 8 of them reported the proportion of outborn babies), and 11 trials did not provide any information, Similarly, 15 studies reported data of participants with only severe or moderate neonatal encephalopathy, 9 studies included those with mild encephalopathy also, but the proportion was <25% of the total, and 15 studies did not report details of severity. Among these 15, data on Apgar score and/or cord blood parameters suggested severe disease in some (Table S2 in the Online Supplementary Document). Thirteen studies did not report any data on Apgar scores or cord blood parameters, whereas 26 studies reported either or both (Table S2 in the Online Supplementary Document). In the absence of individual patient data, it is not possible to account for these factors.
Before initiating this review, we listed the primary outcome as mortality or neurologic disability at the age of 18-24 months, in alignment with previous systematic reviews [1,2], and major trials [1,2,10,26,29,30]. Although the composite outcome provides useful information, we believe that it is skewed by the beneficial effects of TH on neurologic outcomes, masking the lack of statistically significant impact on mortality.
Adverse effects of therapeutic hypothermia were reported in various ways, and at various time points, in several trials. Although these are very important to consider, for making informed decisions (at the practice as well as policy levels), in this systematic review, we focused on evidence of efficacy, and did not examine adverse events.
We expected attrition in trials would bias the results in favor of the intervention, but did not observe this for most outcomes.
Finally, is more research required on therapeutic hypothermia for neonatal encephalopathy? Some experts would argue that more trials should be conducted until an optimal information size is achieved following which, further research can be discontinued. This would be very expensive in terms of time and resources. Instead, we suggest that research in local health care systems in resource-constrained settings, could focus on resolving issues such as which neonates are most likely to benefit from TH, predictors of failure, and of course primary prevention.

CONCLUSIONS
This up-to-date systematic review of randomized controlled trials confirmed that therapeutic hypothermia implemented for neonatal encephalopathy reduces neurologic disability and cerebral palsy in diverse settings. However, it has an unclear effect on neonatal, infantile, and childhood mortality. It also does not impact neonatal seizures, or epilepsy during infancy and childhood. The previously reported reduction in mortality was associated with trials of lower methodological quality, but not substantiated by trials with high(er) quality.